The present invention relates to methods of obtaining hepatocyte lineage cells, intermediate cells, and hepatocytes; the resulting cells; as well as materials for producing the same.
Human induced pluripotent stem (iPS) cells can be generated using four reprogramming factors [Takahashi et al., 2007]. IPS cells have the ability to differentiate into various types of cells and can be applied in regenerative medicine [Hirschi et al., 2014]. Hepatic insufficiency is a fatal condition. If hepatocytes can be produced from iPS cells, they could be used for transplantation in patients with hepatic insufficiency [Chang et al., 2012; van Wenum et al., 2014].
Several protocols on the differentiation of iPS cells to hepatocytes have been reported [DeLaForest et al., 2011; Inamura et al., 2010; Si-Tayeb et al., 2010; Song et al., 2009; Takayama et al., 2012; Zaret et al., 2008]. Most of these apply growth factors sequentially, simulating hepatocyte differentiation in fetal liver [DeLaForest et al., 2011; Si-Tayeb et al., 2010; Song et al., 2009; Takayama et al., 2012]. Another approach is to construct a three-dimensional liver using a combination of iPS cells, human umbilical vascular endothelial cells, and human mesenchymal cells [Takebe et al., 2013]. Only a few protocols have used transcription factors [Inamura et al., 2010; Takayama et al., 2012; Tomizawa et al., 2013].
FOXA1 can open compacted chromatin to initiate transcription, and is called the “pioneering factor” [Xu and Zaret, 2012]. FOXA1 is involved in liver development [Zaret and Carroll, 2011]. FOXA3 binds the promoters of hepatocyte-specific genes, such as AFP and albumin, and upregulates their expression [Costa et al., 2003]. The combination of hepatocyte nuclear factor 1A and FOXA1, FOXA3, or hepatocyte nuclear factor 4A can promote transdifferentiation of human fibroblasts to hepatocyte-like cells [Simeonov and Uppal, 2014]. With another combination of FOXA3, hepatocyte nuclear factor 1A and hepatocyte nuclear factor 4A, human fibroblasts have been transdifferentiated to functional hepatocytes [Hang et al., 2014].
Williams' Medium E (WE) was originally established to isolate hepatocytes from a mixture of fibroblasts for rat primary hepatocyte culture [Pretlow and Williams, 1973]. Drug metabolism is preserved in rat primary hepatocytes cultured in WE [Takeba et al., 2011; Wu et al., 1997]. Gluconeogenesis and urea cycle—hepatocyte-specific functions—are also maintained in WE [Dabos et al., 2004; Farghali et al., 2002]. These findings indicate that WE is suitable for hepatocyte culture without loss of hepatocyte-specific functions.
Transcription factors are involved in liver development [Takebe et al., 2013]. CCAAT/enhancer binding protein alpha (CEBPA) was cloned by Antonson et al [Antonson and Xanthopoulos, 1995]. Hepatocytes deficient in CEBPA have the characteristics of both hepatocytes and bile duct epithelial cells [Tomizawa et al., 1998]. The characteristics of biliary epithelial cells can be enhanced in hepatoblasts with lower expression of CEBPA [Yamasaki et al., 2006]. CEBPA might promote the differentiation of hepatoblasts to mature hepatocytes. CCAAT/enhancer binding protein beta (CEBPB) was cloned by Akira et al [Akira et al., 1990]. CEBPB is involved in the differentiation of hepatocytes [Kheolamai and Dickson, 2009]. A combination of transcription factors is, however, not known to initiate differentiation of iPS cells to hepatocytes.
Accordingly, a need exists for new methods of producing hepatocyte lineage cells.